1. Field of the Invention
The present invention relates to a measuring method and a measuring apparatus that utilize attenuated total reflection, such as a surface plasmon sensor that analyzes the properties of substances, based on the generation of surface plasmon. Particularly, the present invention relates to a measuring method and a measuring apparatus that utilizes attenuated total reflection and employs p-polarized light beams.
2. Description of the Related Art
Surface plasmon sensors are known, as a type of sensor that utilizes attenuated total reflection. In metals, free electrons oscillate in groups to generate compression waves, called plasma waves. The compression waves which are generated at the surface of metals are called surface plasmon, when quantized. Various known surface plasmon sensors utilize a phenomenon, in which the surface plasmons are excited by light waves, to analyze properties of samples. Particularly well known surface plasmon sensors are those of a Kretschmann configuration (as disclosed in Japanese Unexamined Patent Publication No. 6(1994)-167443, for example).
Surface plasmon sensors of the Kretschmann configuration basically comprise: a dielectric block, shaped as a prism, for example; a metal film, formed on one surface of the dielectric block and which is brought into contact with a sample; a light source for emitting a light beam; an optical system for causing the light beam to enter the dielectric block at various angles of incidence so that total internal reflection conditions are satisfied at an interface of the dielectric block and the metal film; a photodetecting means for detecting the intensity of the light beam, which has been totally reflected at the interface; and a measuring means for measuring the state of surface plasmon resonance, based on detection results obtained by the photodetecting means.
In order to obtain various angles of incidence for the light beam, a comparatively thin incident light beam may be caused to impinge upon the interface while changing the angle of incidence. Alternatively, a comparatively thick incident light beam may be caused to impinge upon the interface in the form of convergent light or divergent light, so that the incident light beam includes components impinging upon the interface at various angles. In the former case, the light beam which is reflected from the interface at an angle, which varies as the angle of incidence changes, may be detected by a small photodetector which is moved in synchronization with the change of the angle of incidence, or by an area sensor that extends in the direction coincident with the angles of reflected light. In the latter case, an area sensor, which extends in directions such that all the components of light reflected from the interface at various angles can be detected thereby, may be employed.
In a surface plasmon sensor of the construction described above, when a light beam impinges upon the metal film at a particular angle of incidence θsp greater than or equal to the angle of total internal reflection, evanescent waves having an electric field distribution in a sample which is in contact with the metal film are generated, and surface plasmon is excited at an interface between the metal film and the sample. When the wave vector of the evanescent light is equal to the wave number of the surface plasmon and wave number matching is established, the evanescent waves and the surface plasmon resonate and light energy is transferred to the surface plasmon, whereby the intensity of light reflected in total internal reflection at the interface of the dielectric block and the metal film sharply drops. The sharp intensity drop is generally detected as a dark line by the photodetector.
The aforesaid resonance occurs only when the incident light beam is p-polarized. Accordingly, it is necessary to set the surface plasmon sensor so that the light beam enters the interface as p-polarized light. Alternatively, it is necessary to separate and detect only p-polarized light waves from the light beam, which is totally internally reflected at the interface between the dielectric block and the metal film.
When the wave number of the surface plasmon can be known from the angle of incidence θsp at which the phenomenon of attenuated total reflection (ATR) takes place, the dielectric constant of the sample can be obtained. That is,
            K      sp        ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              wherein Ksp represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the speed of light in a vacuum, and εm and εS respectively represent the dielectric constants of the metal and the sample.
When the dielectric constant εS of the sample is known, the refractive index and the like of the sample can be determined on the basis of a predetermined calibration curve or the like. As a result, properties of the sample related to the refractive index, such as the dielectric constant, can be determined, by determining the angle θsp at which attenuated total reflection occurs (hereinafter, referred to as “attenuated total reflection angle θsp”)
As another type of sensor that utilizes attenuated total reflection, there is known a leaky mode sensor as described in, for instance, “Surface Refracto-sensor using Evanescent Waves: Principles and Instrumentations” by Takayuki Okamoto, Spectrum Researches, Vol. 47, No.1 (1998), pp. 21-23 and pp. 26-27. The leaky mode sensor basically comprises: a dielectric block, shaped as a prism, for example; a cladding layer, formed on one surface of the dielectric block; an optical waveguide layer, which is formed on the cladding layer and which is brought into contact with a sample; a light source for emitting a light beam; an optical system for causing the light beam to enter the dielectric block at various angles of incidence so that total internal reflection conditions are satisfied at an interface of the dielectric block and the cladding layer; a photodetecting means for detecting the intensity of the light beam, which has been totally reflected at the interface; and a measuring means for measuring the state of excitation of a waveguide mode, based on detection results obtained by the photodetecting means
In a leaky mode sensor of the construction described above, when the light beam is caused to impinge upon the cladding layer through the dielectric block at an angle greater than or equal to an angle of total internal reflection, evanescent waves are generated in the optical waveguide layer and an evanescent wave having a particular wave number comes to propagate through the optical waveguide layer in a waveguide mode. When the waveguide mode is thus excited, almost all the incident light which generates the evanescent wave having a particular wave number is taken into the optical waveguide layer and accordingly, the intensity of light reflected in total internal reflection at the interface of the dielectric block and the clad layer sharply drops. Because the wave number of light to be propagated through the optical waveguide layer depends upon the refractive index of the sample on the optical waveguide layer, the refractive index and properties of the sample related to the refractive index can be determined, based on the attenuated total reflection angle θsp.
The aforementioned surface plasmon sensors and leaky mode sensors may be utilized to perform random screening in the field of pharmaceutical manufacture. In random screening, specific substances that bond with a desired sensing substance are sought. In this case, the sensing substance is disposed on the thin film (the metal film in the case of a surface plasmon sensor, and the optical waveguide layer and the cladding layer in the case of a leaky mode sensor). Then, various solutions of test targets (sample liquids) are added to the sensing substance. Each time that a predetermined amount of time passes, the attenuated total internal reflection angle θsp is measured. If the test target binds with the sensing substance, the refractive index of the sensing substance changes over time due to the bond. Accordingly, whether the test target is bonding with the sensing substance, that is, whether the test target is the specific substance that bonds with the sensing substance, can be determined by measuring the attenuated total internal reflection angle θsp at predetermined time intervals, thereby measuring whether the attenuated total reflection angle θsp changes. A combination of an antigen and an antibody is an example of the combination of the specific substance and the sensing substance. Alternatively, a combination of an antibody and another antibody may be the combination of the specific substance and the sensing substance. Measurement regarding whether a rabbit antihuman IgG antibody, as a sensing substance, bonds with an antihuman IgG antibody, as a specific substance, and quantitative analysis of the bond, are specific examples of measurement.
Note that it is not necessary to detect the attenuated total reflection angle θsp itself, in order to measure bonding states between test targets and sensing substances. For example, a test target solution may be added to a sensing substance, then the variation in the attenuated total reflection angle θsp may be measured. The bonding state may be measured, based on the degree of the variation of the attenuated total reflection angle θsp.
Cases in which the attenuated total reflection angle θsp itself is measured, and cases in which the variations in the attenuated total reflection angle θsp are measured after adding the test target solution to the sensing substance have been described. In both of these cases, it is necessary to accurately detect the central position of the dark line, that is, the position of the attenuated total reflection angle θsp, at which the intensity of the light beam totally internally reflected at the interface between the dielectric block and the metal film drops sharply, in order to accurately measure the state of attenuated total reflection. However, it had been difficult to detect the position of the attenuated total reflection angle θsp with high accuracy, due to adverse influences from fluctuations in the light intensity distributions of the light beams themselves, and the like. For this reason, a measuring method and a measuring apparatus have been proposed in Japanese Unexamined Patent Publication No. 7(1995)-159319. This method and apparatus utilize the fact that attenuated total reflection occurs only when an incident light beam is p-polarized light. The method and apparatus separates a light beam, which is totally internally reflected at an interface, into p-polarized light waves and s-polarized light waves. A light intensity distribution that reflects the state of attenuated total reflection is measured utilizing the p-polarized light waves, and the light intensity distribution of the light beam itself (hereinafter, referred to as “reference light intensity distribution”) is measured utilizing the s-polarized light waves. The distribution values of the light intensity distribution that reflects the state of attenuated total reflection, measured by utilizing the p-polarized light waves, are divided by the reference light intensity distribution. Thereby, the influence exerted by fluctuations in the light intensity distribution of the light beam is cancelled out. Accordingly, the position of the attenuated total reflection angle θsp is enabled to be detected with high accuracy.
However, in the above measuring method and measuring apparatus that cancels out the influence exerted by fluctuations in the light intensity distribution of the light beam, to enable highly accurate detection of the position of the attenuated total reflection angle θsp, separating means for separating the totally internally reflected light beam into p-polarized light waves and s-polarized light waves is necessary. This leads to greater size and cost for the apparatus, and causes measurements to be troublesome. Further, conventional measuring methods and measuring apparatuses that utilize attenuated total reflection generally employ light sources that emit p-polarized light waves. However, in the case that the reference light intensity distribution is obtained by utilizing s-polarized light waves as described above, a light source that emits both p-polarized light waves and s-polarized light waves becomes necessary. If this type of light source is employed, the output of the p-polarized light waves is decreased, and measurement accuracy is reduced.